Laboratory Protocols

• Cryotomy
• Histological Staining Techniques
  • Index
• Histological fixatives
• Peroxidase-anti-peroxidase Technique
• Preparation of tissue section adhesives
• Processing tissues for light microscopy (basics) (PDF 1.1mb)
• Processing tissues for light microscopy (detailed)
• Sample collection guidelines
• Setting up the light microscope
• Specimen preparation for electron microscopy

Histological Staining Techniques

Methyl green/pyronin (M.G.P.) for R.N.A. & D.N.A.

Unna, 1902; Pappenheim, 1899.

• Dewax sections, rinse in alcohol, rinse in water.
• Rinse in pH4.8 buffer.
• Stain in M.G.P. solution - 25 minutes.
• Rinse in pH4.8 buffer.
• Rinse in a mixture of equal parts acetone and xylene (or Histoclear).
• Dehydrate in 100% alcohol.
• Rinse in xylene (or Histoclear).
• View microscopically without mounting and repeat the last three steps if necessary.
• Mount in DPX.

Results: 

R.N.A. - (notably in plasma cell cytoplasm), acid mucins - magenta.
D.N.A. - green or purplish green.

M.G.P. solution

9ml 2% aqueous methyl green CI 42590 (if using methyl green CI 42585 it must be *chloroform extracted).
4ml 2% aqueous pyronin Y (or G = gelb, yellow).
23ml acetate buffer pH4.8
14ml glycerol.

Acetate buffer pH4.8

Add 20ml 0.2M acetic acid to 30ml 0.2M sodium acetate.

* Chloroform extraction:  mix equal quantities of stain solution and chloroform in a separating funnel.  Shake them together well, allow the chloroform to settle to the bottom and drain it off.  Repeat this procedure with fresh chloroform until it has no purple tinge.  Dispose of the chloroform and filter the methyl green.

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